胃癌线粒体DNA D-loop区碱基突变的检测分析

目的 研究胃癌患者的癌组织线粒体DNA （mtDNA）D-loop区碱基突变情况,以探讨mtDNA D-loop区碱基突变与胃癌发生发展的相关性.方法 采用聚合酶链反应和DNA测序相结合的方法,对75例胃癌患者癌组织线粒体D-loop 区进行扩增并测序分析.结果 在75例胃癌组织中共有130个位点发生了变化,其中属于基因多态性有101个,基因突变有29个.75例胃癌组织中39例mtDNA D-loop区存在突变,突变率为52%.结论 mtDNA D-loop 区碱基突变可能在胃癌发生发展中发挥重要作用.     

食管癌组织中线粒体DNA突变检测

目的：检测食管癌组织中线粒体DNA(mtDNA)的突变。方法：提取8例食管癌组织的mtDNA；PCR扩增mtDNA的控制区，纯化PCR产物、与T载体连接，转化人肠杆菌，筛选出阳性克隆，用引物T7／SP6做PCR鉴定后测序，与GenRank序列对比。结果：8例标奉mtDNA的控制区共有36个位点发生突变，其中转换32个，颠换4个；同义突变13个，错义突变23个。结论：mtDNA突变很可能在食管癌的发生中发挥一定作用。     

胃癌组织线粒体DNA D-loop区的突变及其意义

目的研究胃癌组织中线粒体DNA(mtDNA)D-loop区突变情况及其在肿瘤发生和发展中的作用。方法选择17例胃癌及相应正常胃黏膜组织,应用聚合酶链反应(PCR)对其mtDNA D-loop区进行扩增并测序,将测序结果与线粒体文库中的Revised Cambridge Reference Sequence(rCRS)进行对比分析。结果 17例胃癌组织中共发现mtDNA D-loop区存在175个多态性变异,其中8个(4.6%)为新发现的变异。8例(47.1%)胃癌组织中共发现13次突变;突变热点集中在HV1(23.1%)和HV2(53.9%),并且HV2较HV1更易突变。mtDNA D-loop区突变率与胃癌分化程度、浸润深度、有无淋巴结转移、病人性别和年龄无关(P>0.05)。结论 mtDNA D-loop区尤其是其中的HV1及HV2是一个具有高度多态性和突变性的区域,在胃癌中突变率较高。     

骨髓增生异常综合征患者线粒体DNA D-loop区突变研究

目的检测骨髓增生异常综合征（myelodysplastic syndrome, MDS）患者线粒体DNA D-loop区的突变。方法提取42例MDS患者骨髓以及口腔上皮组织的线粒体DNA，对线粒体DNA D-loop区两个高变区（HV1和HV2）进行PCR扩增，通过直接测序的方法对PCR产物的基因序列进行检测。结果在42例MDS患者中有6例患者出现突变，突变率为14.29%。突变位点19个，10个位于HV1区，9个在HV2区，其中包括5个微卫星不稳定，其余为A、G或者C、T之间的碱基置换。结论MDS存在线粒体DNA D-loop区的突变，可能在疾病的发生发展中起重要作用。     

人大肠癌线粒体DNA重组质粒的构建及对NIH3T3细胞的转染

目的: 构建pcDNA3. 1( )-mtDNA真核表达重组体,并导入NIH3T3细胞.方法: 提取大肠癌细胞株(SW480,Lovo,HT29)mtDNA,扩增D-loop区,产物用DNA自动测序法进行序列分析.利用DNA重组技术将其定向插人真核表达质粒pcDNA3. 1( ),并用脂质体法导入NIH3T3细胞.结果: 大肠癌细胞株SW480,Lovo,HT29细胞mtDNA D-loop分别有10,9,8个突变位点.成功克隆1119 kb的mtDNA D-loop区至表达质粒pcDNA3. 1( ),并导入NIH3T3细胞中.结论: 成功构建了pcDNA3. 1( )-mtDNA真核表达重组体,并成功导入NIH3T3细胞中.     

人大肠癌线粒体DNA重组质粒的构建

目的了解大肠癌细胞株（SW480，LoVo，HT29）线粒体DNA的突变，克隆突变的大肠癌线粒体DNA（mtDNA）基因，构建pcDNA3．1（＋）-mtDNA真核表达重组体，并导入NIH3T3细胞，以探讨线粒体基因突变与肿瘤发生的关系。方法提取大肠癌细胞株（SW480，LoVo，HT29）mtDNA，扩增D-LOOP区，产物用DNA自动测序法进行序列分析。利用DNA重组技术将其定向插人真核表达质粒pcDNA3．1（＋），并用脂质体法导人NIH3T3细胞。结果检测出大肠癌细胞株SW480、LoVo、HT29细胞mtDNAD—LOOP分别有10、9、8个突变位点。成功克隆1119bp的mtDNAD—LOOP区至表达质粒pcDNA3．1（＋），并导入NIH3T3细胞中。结论线粒体DNAD-LOOP区是一个具有高度多态性和突变性的区域，在大肠癌细胞株中突变率较高。     

Single Nucleotide Polymorphisms （SNPs） and Variable Number Tandem Repeats （VNTRs） in mtDNA D-loop and CO Ⅱ- tRNA^Lys Intergenic Region with PCOS

Objective To explore the relationships of variations in D-loop and COII-tRNA Lys intergenic region in mtDNA with PCOS. Methods A total of 77 PCOS and 45 non-PCOS patients were enrolled, whose D-loop and COII-tRNA Lys intergenic region in mtDNA were amplified and sequenced; sexual hormone assay, oral glucose tolerance test (OGTT) and insulin releasing test were carried out. Then variations found in mtDNA were compared between the two groups, the correlations between variations and clinical indexes were analyzed in all subjects. Results Nucleotide variations found in mtDNA were not different between the two groups, but the mutation of 16 094T/C was found associated with the serum levels of T and fasting insulin; (303-317)Cn TCn associated with the serum levels of A and LH; 195C/T with A level and 491T/C with LH level; (8 272-8 289)(ACCCCCTCT)n was associated with the serum level of 1 h glucose. Conclusion Noncoding region mutations in mtDNA perhaps associate with PCOS clinical symptoms and involve in PCOS development.     

人大肠癌线粒体DNA重组质粒的构建

目的 了解大肠癌细胞株(SW480,LoVo,HT29)线粒体DNA的突变,克隆突变的大肠癌线粒体DNA(mtDNA)基因,构建pcDNA3.I(+)-mtDNA真核表达重组体,并导入NIH3T3细胞,以探讨线粒体基因突变与肿瘤发生的关系。方法 提取大肠癌细胞株(SW480,LoVo,HT29)mtDNA,扩增D-LOOP区,产物用DNA自动测序法进行序列分析。利用DNA重组技术将其定向插人真核表达质粒pcDNA3.1(+),并用脂质体法导人NIH3T3细胞。结果 检测出大肠癌细胞株SW480、LoVo、HT29细胞mtDNAD-LOOP分别有10、9、8个突变位点。成功克隆1119bp的mtDNAD-LOOP区至表达质粒pcDNA3.1(+),并导入NIH3T3细胞中。结论 线粒体DNAD-LOOP区是一个具有高度多态性和突变性的区域,在大肠癌细胞株中突变率较高。     

高低转移小鼠肝癌细胞线粒体DNA遗传变异的研究

目的 检测和分析转移特性不同的两个小鼠肝癌细胞亚系线粒体DNA(mtDNA)的遗传变异,探讨线粒体DNA遗传改变与肿瘤发生发展的关系.方法 PCR-RFLP和序列测定技术.结果 对mtDNA的tRNA Ile GlN Met基因和ND3基因以及D-loop片段进行的扩增和限制性片段长度多态性分析结果 显示,无扩增片段长度呈多态性,且这两个肝癌细胞系mtDNA的所有限制性片段方式和大小完全一致.序列测定发现,这两个肝癌细胞系在线粒体DNA的 D-loop区存在序列差异.结论 mtDNA 非编码区内的遗传改变,反映了肿瘤发生发展过程中环境和遗传因素的影响,有可能与肿瘤细胞的恶性表型有关.     

Delta mtDNA4977 is more common in non-tumoral cells from gastric cancer sample

BACKGROUND: The aim of this study was to determine the frequency of delta mtDNA4977 in tumoral cells as compared with adjacent normal cells in gastric cancer. METHODS: In order to investigate whether a high incidence of mutation exists in mitochondrial DNA of gastric cancer tissues, we screened one of common region of the mitochondrial genome by PCR amplification and Southern blot followed by DNA sequence analysis. DNA isolated from these cells was used to amplify hypervariable regions ATPase8/6, COXIII, ND3, ND4 and ND5 of delta mtDNA4977. RESULTS: In 107 cancer patients, delta mtDNA4977 was detected in 6 cases (5.60%) of the tumoral tissues and 18 cases (16.82%) of the non-tumoral tissues that were adjacent to the tumors. Levels of delta mtDNA4977 deletions were found to be more in non-tumoral tissues than in adjacent tumoral tissues. There was no correlation of patients with certain clinical parameters like age, sex, tumor location and tumor size; however, there was an obvious relationship with intestinal-type of gastric cancer. CONCLUSIONS: Unknown genetic aspects, ambiguous environmental factors and reactive oxygen species (ROS) can cause the delta mtDNA4977 mutation rate to be increased in gastric cancer. The results suggest that percentage level of delta mtDNA4977 is less common and intolerable in tumoral tissue, probably because of high metabolism and ROS generation. We supposed that the cells initially had delta mtDNA4977 transform to tumoral cells and the existed deletion conferred metabolic disadvantage; thus, cells containing such a mtDNA deletion would be overgrown by other cancer cells without this mtDNA deletion. As a result, the presence of delta mtDNA4977 will be low in tumoral cells.     

宣威地区非小细胞肺癌患者线粒体DNA突变与多态性研究

【目的】寻找宣威地区肺癌患者肺组织中线粒体DNA突变和多态性情况,为进一步研究宣威地区女性肺癌高发机制提供参考。【方法】征集28例宣威籍肺癌患者,收集癌组织和相应癌旁正常肺组织,提取线粒体DNA ,实时荧光定量PCR和直接测序方法检测线粒体DNA突变和多态性改变。【结果】28例肺癌组织样本同对应癌旁组织比较,有21例（75．0％）发生mtDNA突变,15例有多种突变,突变发生在DLOOP 区,以及呼吸链编码区等区域。突变与患者年龄、性别,及组织学类型无明显联系。28例肺癌组织、相应癌旁组织同M t-DNA剑桥序列比较,有3例有mtDNA多态性改变。【结论】宣威肺癌患者线粒体DNA存在特异性的突变位点,以及多态性位点,可能和当地特殊的燃煤污染暴露及遗传易感性有关。     

Mitochondrial D-loop variation in leber hereditary neuropathy patients harboring primary G11778A, G3460A, T14484C mutations: J and W haplogroups as high-risk factors

BACKGROUND: Leber hereditary optic neuropathy (LHON) is a maternally inherited form of retinal ganglion cell degeneration leading to optic atrophy in young adults. It is caused by three primary point mutations including G11778A, G3460A, and T14484C in the mitochondrial genome. These three mutations account for the majority of LHON cases and affect genes that encode for different subunits of mitochondrial complex I. Mitochondrial DNA (mtDNA) has a non-coding region at the displacement loop (D-loop) that contains two hypervariable segments (HVS-I and HVS-II) with high polymorphism. METHODS: To investigate any possible association between LHON primary mutations and mtDNA haplogroups (hg), the nucleotide sequence of the HVS-I region of mtDNA was determined in 30 unrelated Iranian patients with LHON harboring one of the primary mutations and 100 normal controls with the same ethnicity. DNA was extracted from the peripheral blood after having obtained informed consent. The nucleotide sequence of HVS-I (np 16,024-16,383) was directly determined. RESULTS: Our analysis revealed a relatively high proportion of haplogroup J in LHON patients (53.3%) compared to normal controls (20%). In addition, a slightly significant increase of normal controls of haplogroup L has been confirmed (14% in normal controls vs. 0% in LHON patients at p = 0.03), whereas other haplogroups did not show contribution to LHON contingency. CONCLUSIONS: The analysis presented here provides evidence that there is an association between G11778A and G3460A with haplogroup J (including J1 and J2) and W, respectively. Therefore, we hypothesize that mtDNA haplogroups J (J1 and J2) and W might act as predisposing haplotypes, increasing penetrance of LHON disease.     

几种肿瘤细胞株线粒体DNA多态性

目的了解和分析线粒体DNA突变与肿瘤发生发展的关系.方法应用PCR-RFLP和PCR-SSCP技术对Lewis肺癌等肿瘤细胞株mtDNA的D-loop,ND3 and tRNAMet Glu Ile基因片段进行了分析.结果mtDNA编码区的tRNAMet glu Ile及ND1基因核酸片段经HaeⅢ等16种内切酶消化后,这些肿瘤细胞株表现出完全相同的酶切图谱.而D-loop片段,小鼠肿瘤Hca-F出现了与对照小鼠不同的酶切方式;用PCR-SSCP分析方法对这些肿瘤细胞株mtDNA的D-loop的5'及3'端作进一步分析,在研究的6个肿瘤中,有4个在mtDNA非编码区上存在着突变.结论mtDNA突变在肿瘤的发生发展中可能起一定作用.     

人肝癌SMMC-7721细胞株部分线粒体基因表达的研究

目的:观察人肝癌SMMC-7721细胞株部分线粒体DNA(mtDNA)基因表达的情况.方法:根据人线粒体DNA(mtDNA)基因序列,利用primer premier 5.0生物软件设计了4对PCR引物,分别是扩增D-loop区、ND6、ATPase8-ATPase6、16sRNA基因,采用RT-PCR方法对SMMC-7721细胞线粒体DNA上D-loop区及其他3个基因的表达进行了检测,并与其在正常人肝细胞株(L02)线粒体中的表达作了比较.结果:我们发现,与正常人肝细胞株(L02)相比,在SMMC-7721细胞中,D-loop区、ND6、ATPase8-ATPase6基因表达总体是增高的,16sRNA基因表达基本不变,同时在同一基因两条链(重链和轻链)之间的表达也存在差异.结论:我们认为,由于线粒体DNA编码的多肽均是氧化磷酸化酶复合物的亚单位,这些基因表达的异常可能造成细胞对氧利用障碍,细胞能量产生减少,成为造成SMMC-7721细胞主要以糖酵解的方式提供能量的重要原因之一.     

Mutations of mitochondrion DNA in mouse tumors

Objective： To ascertain the variations of mitochondrion DNA （mtDNA） in mouse tumors and to inquire into the relationship between mutations of mtDNA and carcinogenesis Methods： The variations of D-loop, ND3 and tRNA^Met＋Glu＋Ile gene fragments of mtDNA from six tumor cell lines of mice were analyzed by PCR technology with restriction fragment length polymorphism analysis （polymerase chain reaction-restriction fragment length polymorphism, PCR-RFLP） and single strand conformation polymorphism analysis （SSCP-PCR） method. Results： ND3 and tRNA^Met＋Glu＋Ile gene fragments ofmtDNA from the tumors showed no variation in 27 endonuclease sites; D-loop ofmtDNA from Hca-F had an additional endonuclease sites of Hinf I in contrast to that of the inbred mouse. Deeply analyzed by PCR-SSCP, the D-loop ofmtDNA was found to possess mutations in 4 of 6 tumors. Conclusion： D-loop is the hot spot of tumor mtDNA mutation which can act as contributors to the carcinogenic     

Tumoral cell mtDNA approximately 8.9 kb deletion is more common than other deletions in gastric cancer

BACKGROUND: The aim of the study was to clarify the role of deletion of mitochondrial DNA (mtDNA) in gastric carcinogenesis and to determine prevalence of mitochondrial deletions in different regions of tumoral tissue in comparison with adjacent non-tumoral tissue in gastric cancer. METHODS: In order to investigate whether a high incidence of mutations exists in mtDNA of gastric cancer tissues, we screened five regions of the mitochondrial genome by PCR amplification, Southern blot and DNA sequence analysis. RESULTS: Of 71 cancer patients, the approximately 8.9 kb deletion was detected among different deletions in 9 cases (12.67%) of the tumoral tissues and 1 case (1.40%) in non-tumoral tissues that were adjacent to the tumors. Level of the 8.9 kb deletion has been found to be more than other deletions in tumoral tissues. CONCLUSIONS: The approximately 8.9 kb deletion has an obvious correlation with age and histological type. These data suggest that the approximately 8.9 kb deletion in mtDNA may play an important role in gastric carcinogenesis.     

颞下颌关节骨关节病髁突软骨细胞线粒体DNA突变的实验研究

目的:探讨颞下颌关节骨关节病髁突软骨细胞线粒体DNA突变及其意义.方法:去除左侧部分关节盘建立大鼠颞下颌关节骨关节病模型,培养骨关节病(术后3月)和正常髁突软骨细胞.采用32对引物,使用PCR技术部分重叠扩增全长线粒体DNA,并将PCR产物进行时间温度梯度电泳,对电泳条带与正常有差异的PCR产物进行测序.结果:在35个具有异质性突变特点的PCR产物中,发现42个异质性突变,tRNA和D-loop具有高突变率.结论:颞下颌关节骨关节病髁突软骨细胞线粒体DNA存在突变.     

胰腺癌线粒体DNA突变研究

目的研究线粒体DNA（mtDNA）突变在胰腺癌发病中的作用。方法用PCR与直接测序相结合的方法,对比分析2株胰腺癌细胞株（SW1990、JF-305）和1株原代培养的正常胰腺细胞mtDNA D环区的突变位点。结果 2株胰腺癌细胞和1株正常的胰腺细胞的mtDNA D-loop区均存在不同程度的点突变,SW1990共检测到8个突变位点,JF-305共检测9个突变位点。其中73位A-G、16223位C-T和16358位C-T这3个突变位点在2株癌细胞和正常胰腺细胞中均检测到,考虑为多态性变化;16211位C-T和16311位T-C2个相同的突变位点在2株胰腺癌细胞中均检测到,考虑为特征性突变。结论胰腺癌细胞mtDNAD环区具有多态性和突变性,其突变可能与胰腺癌的发生、发展密切相关。     

突变的大肠癌线粒体DNA转染NIH3T3及LST细胞的研究

日的了解大肠癌细胞株（SW480，LOVO，HT29）线粒体DNA的突变，克隆突变的大肠癌线粒体DNA（mtDNA）基因，构建pcDNA3．1（＋）-mtDNA真核表达重组体，并导人NIH3T3及LST细胞。以探讨线粒体基因突变与肿瘤发生的关系。方法提取大肠癌细胞株（SW480，LOVO,HT29）mtDNA，扩增D-LOOP区，产物用DNA自动测序法进行序列分析。利用DNA重组技术将其定向插人真核表达质粒pcDNA3．1（＋）。并用脂质体法导人NIH3T3及LST细胞。用MitoCapture Mitochondrial Apoptosis Detection Kit试剂盒染色后用流式细胞仪及荧光显微镜检测转染细胞的凋亡情况。扩增并测序分析转染细胞的D-LOOP区突变特点。结果检测出大肠癌细胞株SW480、LOVO和HT29细胞mtDNAD-LOOP分别有10、9和8个突变位点。转染前后，各组间细胞凋亡无明显变化。转染细胞的核基因组可扩增出目的基因及Neo基因。4株NIH3T3转染细胞mtDNA D-环区分别检测到9、11、8和4个突变点，并相应有3、4、3和2个多态性变化。结论转染突变的大肠癌细胞mtDNA后转染细胞的mtDNA均可发生多处的突变位点；通过转染后突变的外源性的mtDNA可以整合到核基因组内；突变的mtDNA转染LST细胞及NIH3T3细胞后。不影响转染细胞的凋亡改变；mtDNA的突变可能通过影响体细胞mtDNA的突变和通过外源性mtDNA在核内的整合从而影响癌基因或抑癌基因的表达异常，从而参与肿瘤的发生发展。     

线粒体DNA D-loop区多态性与肝癌预后的研究

目的通过对线粒体DNA D-loop区的碱基测序,了解其多态性及突变情况,并探讨其与肝癌发病机率及预后的关系。方法提取49个乙型肝炎后肝癌病人的肝癌组织,癌旁组织和血液中的线粒体DNA,用测序法测定线粒体DNA D-loop区的碱基序列,了解其单核苷酸多态性和变异情况,并分析其与肝癌发病率及2年生存率的关系。结果通过观察发现,肝癌病mt DNA突变情况与肝癌预后无明确相关,而1个SNP位点(核苷酸150C/T)与肝癌的生存期之间存在统计学上的显著差异。经COX回归分析,发现150位点为肝癌预后的独立危险因素;150C的患者生存期显著短于150T的生存期(相对危险度,0.246;95%CI,0.070-0.861;P=0.028)。结论通过分析在线粒体DNAD-loop区的多态性,可以帮助筛别预后不良的肝癌患者。